ErbB2 overexpression is found in ~ 30% of human breast cancers and many other cancer types. ErbB2 overexpression leads to a very aggressive, highly metastatic breast cancer phenotype and poor patient survival. The ErbB2-targeting antibody, trastuzumab (Herceptin) has shown remarkable therapeutic efficacy in some patients with ErbB2-overexpressing breast cancer. The overall trastuzumab response rate, however, is limited and the causes of trastuzumab resistance are poorly understood. We have recently found that a rapid PTEN activation in ErbB2-overexpressing breast cancer cells by trastuzumab contributes to the anti-tumor activity of trastuzumab. Reducing PTEN expression in ErbB2-overexpressing human breast cancer cells by PTEN antisense oligonucleotides (PTEN-AS) conferred trastuzumab resistance in vitro and in xenograft models. More importantly, among a small cohort of patients having ErbB2- overexpressing breast cancers treated with trastuzumab plus taxanes, those with PTEN-deficient cancers had significantly worse responses to trastuzumab-based therapy than those with normal PTEN (p<0.01). We also found that PI3K inhibitors LY294002 and wortmannin rescued trastuzumab resistance induced by PTEN loss in vitro and in xenograft models. Thus, we hypothesize that a) PTEN deficiency in an ErbB2- overexpressing breast tumor is a sensitive and specific predictor for trastuzumab resistance, and b) Combination therapies with trastuzumab and agents targeting PI3K pathways can be developed to overcome the trastuzumab resistance mediated by PTEN loss. To test our hypotheses, we will first determine whether PTEN deficiency predicts trastuzumab resistance in a large-scale (200 patients) retrospective study (Aim 1). To investigate effective combination therapies that overcome the PTEN loss mediated trastuzumab resistance, we will establish mice models bearing PTEN-deficient and ErbB2- overexpressing mammary tumors and mammary epithelial cell (MEC) strains from the tumors (Aim 2). We will then test therapeutics that reverses PTEN deficiency-mediated trastuzumab resistance in vitro and in vivo using the mice models and the MEC strains (Aim 3). Finally, we will test whether combination therapy of trastuzumab and inhibitors of the PI3K signaling pathway overcome the PTEN loss-mediated trastuzumab resistance in patients (Aim 4). PTEN deficiency has been found in approximately 50% of breast cancer patients, our proposed studies will define the role of this deficiency in trastuzumab resistance. More importantly, the proposed studies will investigate novel treatment regimens for trastuzumab-resistant patients and may provide new therapeutic options that dramatically improve the clinical outcomes for these patients.